Method for producing astaxanthin by fermentation

ABSTRACT

An object of the present invention is to provide a method for microbiologically producing astaxanthin of high concentration at low cost while suppressing production of canthaxanthin. Specifically, the present invention relates to a method for producing carotenoids including astaxanthin comprising culturing a bacterium that concurrently produces astaxanthin and canthaxanthin in a medium containing biotin, wherein a ratio of concentration of produced canthaxanthin to concentration of produced astaxanthin in a culture product after the end of culture in the medium is lower than that in a culture product after the end of culture in a biotin-free medium.

RELATED APPLICATIONS

This application is a national stage application filed under 35 USC §371of PCT/JP2011/056033, filed Mar. 15, 2011, which claims the benefit ofJapanese Patent Application No. 2010-057321, filed Mar. 15, 2010. Theentire contents of each of the above-mentioned applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a method for producing carotenoids,including astaxanthin, by microorganism fermentation.

BACKGROUND ART

Carotenoids are natural pigments that are useful as feed additives, foodadditives, pharmaceutical agents, and the like. Examples of carotenoidsinclude astaxanthin, canthaxanthin, zeaxanthin, β-cryptoxanthin,lycopene, β-carotene, phoenicoxanthin, adonixanthin, echinenone,asteroidenone, and 3-hydroxyechinenone.

Among carotenoids, astaxanthin is useful as feed additives, for example,as a body color-improving agent for cultivated fishes such as salmon,trout, and sea bream or an egg yolk color-improving agent for poultry.Moreover, astaxanthin is highly valuable in the industries as safenatural food additives and health food materials.

Similar to astaxanthin, adonixanthin and phoenicoxanthin are alsoexpected to be used as feed additives, food additives, pharmaceuticalagents, and the like once industrial production methods thereof areestablished. Furthermore, β-carotene is used as feed additives, foodadditives, pharmaceutical agents, and the like; canthaxanthin is used asfeed additives, food additives, cosmetics, and the like; and zeaxanthinis used as food additives, feed additives, and the like. In addition,other carotenoids such as lycopene, echinenone, β-cryptoxanthin,3-hydroxyechinenone, and asteroidenone are also expected to be used asfeed additives, food materials, and the like. Known methods forproducing these carotenoids include chemical synthesis methods, methodsof extraction from natural sources, and production methods usingmicroorganisms.

As methods for chemically synthesizing astaxanthin, a method utilizingconversion of β-carotene (Non-Patent Document 1) and a method employingsynthesis from C15 phosphonium salt (Non-Patent Document 2) are known.Astaxanthin produced by such chemical synthesis methods is commerciallyavailable as a feed additive. In addition, since astaxanthin is presentin fishes such as sea bream and salmon as well as crustaceans such asshrimp, crab, and krill, it may also be extracted therefrom.

Methods that have been reported for producing astaxanthin usingmicroorganisms include a culture method using green alga Haematococcuspluvialis (Patent Document 1), a fermentation method using red yeastPhaffia rhodozyma (Patent Document 2), and a fermentation method using abacterium belonging to the genus Paracoccus (hereinafter occasionallyreferred to as a “Paracoccus bacterium”).

Examples of astaxanthin-producing Paracoccus bacteria include strainsE-396 and A-581-1 (Patent Document 3 and Non-Patent Document 3).Examples of other astaxanthin-producing Paracoccus bacteria includeParacoccus marcusii strain MH1 (Patent Document 4), Paracoccushaeundaensis strain BC74171 (Non-Patent Document 4), Paracoccusbacterial strain N-81106 (Patent Document 5), and Paracoccus sp. strainPC-1 (Patent Document 6).

There have been several problems concerning the above-mentionedcarotenoid production methods. For example, chemical synthesis methodshave unfavorable impression on consumers from a safety perspective.Extraction from natural sources such as shrimp and crab is associatedwith high production costs. In addition, production using a green algaor yeast results in low productivity and has difficulty in extraction ofa carotenoid therefrom due to strong cell walls thereof.

Meanwhile, bacteria belonging to the genus Paracoccus are advantageousin that proliferation rates thereof are fast, carotenoid productivitiesthereof are high, and carotenoids can readily be extracted therefrom,etc. Several methods for culturing such bacteria have been reported. Forexample, Patent Document 7 discloses a method characterized by adding aniron salt during culture. Patent Document 8 discloses a methodcharacterized by restricting the carbon source concentration. However,such culture methods are problematic in that large amounts ofcanthaxanthin accumulate during production of astaxanthin.

Canthaxanthin is a useful feed additive for improving the color tone ofsalmon meat or hen egg yolk, while the ADI (acceptable daily intake)thereof is limited to 0.03 mg/kg of body weight and the upper limits ofthe amounts of canthaxanthin acceptable to be added to feed arestipulated as 25 mg/kg and 8 mg/kg for salmon and laying hens,respectively, in Europe (Non-Patent Document 5). Thus, it is necessaryto control the canthaxanthin content at a low level when producingastaxanthin using a microorganism. Patent Document 9 discloses a methodwherein canthaxanthin production is reduced by controlling the dissolvedoxygen concentration. However, such a method also results in significantreduction in the concentration of produced astaxanthin and thus is notpractical in terms of production cost.

CITATION LIST Patent Literatures

-   Patent Document 1: JP Patent Application Laid-open Publication No.    2007-97584 A-   Patent Document 2: JP Patent Application Laid-open Publication No.    11-69969 A (1999)-   Patent Document 3: JP Patent Application Laid-open Publication No.    7-79796 A (1995)-   Patent Document 4: JP Patent Application Laid-open Publication No.    2001-512030 A-   Patent Document 5: JP Patent Application Laid-open Publication No.    2007-244205 A-   Patent Document 6: WO 2005/118812-   Patent Document 7: JP Patent Application Laid-open Publication No.    2007-143492 A-   Patent Document 8: JP Patent Application Laid-open Publication No.    2008-167665 A-   Patent Document 9: JP Patent Application Laid-open Publication No.    2001-352995 A

Non-Patent Literatures

-   Non-Patent Document 1: Erich Widmer et al., “Pure Appl. Chem.,”    1985, vol. 57, pp. 741-752-   Non-Patent Document 2: Erich Widmer et al., “Hely. Chim. Acta,”    1981, vol. 64, pp. 2436-2446-   Non-Patent Document 3: Akira Tsubokura et al., “International    Journal of Systematic Bacteriology,” 1999, vol. 49, pp. 277-282-   Non-Patent Document 4: Jae Hyung Lee et al., “International Journal    of Systematic and Evolutionary Microbiology,” 2004, vol. 54, pp.    1699-1702-   Non-Patent Document 5: Official Journal of the European Communities    L 22/28-30, Jan. 25, 2003

SUMMARY OF INVENTION Problems to be Solved by the Invention

The present invention has been made in view of the above circumstances.An object of the present invention is to provide a method formicrobiologically producing astaxanthin of high concentration at lowcost while suppressing production of canthaxanthin.

Means for Solving the Problems

As a result of intensive studies in order to achieve the above object,the present inventors have found that production of highly concentratedastaxanthin can be achieved while maintaining the concentration ofproduced canthaxanthin at a low level by adding biotin to a mediumduring culture of a bacterium that concurrently produces astaxanthin andcanthaxanthin. This has led to the completion of the present invention.

The present invention encompasses the followings.

-   (1) A method for producing carotenoids including astaxanthin    comprising culturing a bacterium that concurrently produces    astaxanthin and canthaxanthin in a medium containing biotin, wherein    a ratio of concentration of produced canthaxanthin to concentration    of produced astaxanthin in a culture product after the end of    culture in the medium is lower than that in a culture product after    the end of culture in a biotin-free medium.-   (2) The method according to (1), wherein a concentration of biotin    in the medium is 0.001 mg/L to 50 mg/L.-   (3) The method according to (1), wherein the ratio of concentration    of produced canthaxanthin to concentration of produced astaxanthin    in the culture product after the end of culture is 25% by mass or    less.-   (4) The method according to (1), wherein a concentration of produced    gluconic acid in the culture product after the end of culture is 30    g/L or less.-   (5) The method according to (1), wherein a dissolved oxygen    concentration in the culture product is controlled at 1 ppm or more    during culture.-   (6) The method according to (1), wherein a poly-β-hydroxybutyrate    (hereinafter referred to as “PHB”) content in the culture product    after the end of culture based on dry cells is 30% by mass or less.-   (7) The method according to (1), wherein a dissolved oxygen    concentration in the culture product is controlled at 1 ppm or more    during culture, and the ratio of concentration of produced    canthaxanthin to concentration of produced astaxanthin in the    culture product after the end of culture is 25% by mass or less.-   (8) The method according to (1), wherein a dissolved oxygen    concentration in the culture product is controlled at 2 ppm or more    during culture, and the ratio of concentration of produced    canthaxanthin to concentration of produced astaxanthin in the    culture product after the end of culture is 8% by mass or less.-   (9) The method according to (1), wherein a dissolved oxygen    concentration in the culture product is controlled to increase in a    stepwise or continuous manner during culture.-   (10) The method according to (1), wherein an initial dissolved    oxygen concentration in the culture product is controlled at 1 to    2.5 ppm in the intermediate phase of culture and the dissolved    oxygen concentration is increased in a stepwise or continuous    manner, and the ratio of concentration of produced canthaxanthin to    concentration of produced astaxanthin in the culture product after    the end of culture is 25% by mass or less.-   (11) The method according to (1), wherein an initial dissolved    oxygen concentration in the culture product is controlled at 2 to    3.5 ppm in the intermediate phase of culture and the dissolved    oxygen concentration is increased in a stepwise or continuous    manner, and the ratio of concentration of produced canthaxanthin to    concentration of produced astaxanthin in the culture product after    the end of culture is 8% by mass or less.-   (12) The method according to (1), wherein the bacterium is a    bacterium belonging to the genus Paracoccus.-   (13) The method according to (1), wherein the bacterium is of a    mutant strain having lowered PHB producing ability.-   (14) The method according to (1), wherein the bacterium is of a    mutant strain having lowered gluconic acid producing ability.-   (15) The method according to (1), wherein the bacterium is a    bacterium in which the nucleotide sequence of DNA corresponding to    16S ribosomal RNA is substantially homologous to the nucleotide    sequence shown in SEQ ID NO: 1.-   (16) The method according to (15), wherein the bacterium is of    strain E-396 (FERM BP-4283) or A-581-1 (FERM BP-4671) or a mutant    strain thereof.-   (17) A carotenoid composition for feed comprising carotenoids    including astaxanthin produced by the method according to (1),    wherein the ratio of concentration of canthaxanthin to concentration    of astaxanthin in the produced carotenoids is 25% by mass or less.-   (18) The carotenoid composition for feed according to (17), wherein    a PHB content in the produced carotenoids including astaxanthin is    30% by mass or less.-   (19) A carotenoid composition for food comprising carotenoids    including astaxanthin produced by the method according to (1),    wherein the ratio of concentration of canthaxanthin to concentration    of astaxanthin in the produced carotenoids is 8% by mass or less.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2010-057321, which is a priority document of the present application.

Effects of the Invention

According to the present invention, astaxanthin of high concentrationcan be microbiologically produced at low cost while keeping thecanthaxanthin concentration at a low level. Carotenoids produced by thepresent invention are useful as feed and food materials.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail. Thescope of the present invention should not be limited by the descriptionsbelow and may appropriately be modified and carried out apart from thefollowing illustrative embodiments without departing from the spirit ofthe present invention.

The present invention relates to a method for producing carotenoids,including astaxanthin, by culturing a bacterium that concurrentlyproduces astaxanthin and canthaxanthin (hereinafter occasionallyreferred to as a “carotenoid-producing bacterium” or“astaxanthin-producing bacterium”) in a medium containing biotin(hereinafter referred to as “the method of the present invention”).According to the method of the present invention, a ratio ofconcentration of produced canthaxanthin to concentration of producedastaxanthin in a culture product after the end of culture in the mediumcontaining biotin is lower than that in a culture product obtained usinga similar carotenoid-producing bacterium after the end of culture in abiotin-free medium. According to the method of the present invention, itbecomes possible to produce astaxanthin of high concentration at lowcost by adding biotin to a medium while suppressing the concentration ofproduced canthaxanthin.

A bacterium used in the method of the present invention is not limitedas long as it concurrently produces astaxanthin and canthaxanthin.However, bacteria belonging to the genus Paracoccus are preferably used.Of the bacteria belonging to the genus Paracoccus, Paracoccuscarotinifaciens, Paracoccus marcusii, and Paracoccus haeundaensis arepreferably used, and Paracoccus carotinifaciens is particularlypreferably used. Specific examples of bacterial strains belonging to thegenus Paracoccus include Paracoccus carotinifaciens strain E-396 (FERMBP-4283) and Paracoccus bacterial strain A-581-1 (FERM BP-4671) (PatentDocument 3 and Non-Patent Document 3). These bacterial strains also canbe preferably used in the method of the present invention.

As a carotenoid-producing bacterium, a bacterium in which the nucleotidesequence of DNA corresponding to 16S ribosomal RNA is substantiallyhomologous to the nucleotide sequence of E-396 shown in SEQ ID NO: 1 ispreferably used. The phrase “substantially homologous” as used hereinmeans that homology between nucleotide sequences is preferably 95% ormore, more preferably 96% or more, further preferably 97% or more,particularly preferably 98% or more, and most preferably 99% or more inconsideration of error frequency and the like in DNA sequencing.Homology can be determined using, for example, the Clustal W geneanalysis software.

The phrase “the nucleotide sequence of DNA corresponding to 16Sribosomal RNA” means a nucleotide sequence obtained by substituting U(uracil) in the nucleotide sequence of 16S ribosomal RNA with T(thymine).

Classification of microorganisms based on the homology of the nucleotidesequence of 16S ribosomal RNA is recently becoming the mainstream. Sinceconventional classification of microorganisms is based on conventionallyknown mycological properties such as mobility, auxotrophy, and sugarutilization, microorganisms may incorrectly be classified when therehappens a change in the characteristics due to spontaneous mutation orthe like. On the other hand, the nucleotide sequence of 16S ribosomalRNA is fairly genetically stable, thereby classification based onhomology thereof greatly improves reliability of the classification ascompared to the conventional classification methods.

Homologies between the nucleotide sequence of 16S ribosomal RNA ofParacoccus carotinifaciens strain E-396 and the nucleotide sequences of16S ribosomal RNAs of other carotenoid-producing bacteria, i.e.,Paracoccus marcusii strain DSM 11574 (International Journal ofSystematic Bacteriology (1998), 48, 543-548), Paracoccus bacterialstrain N-81106 (Patent Document 5), Paracoccus haeundaensis strain BC74171 (Non-Patent Document 4), Paracoccus bacterial strain A-581-1, andParacoccus sp. strain PC-1 (Patent Document 6) are 99.7%, 99.7%, 99.6%,99.4%, and 95.4%, respectively, showing that these strains are extremelyclose strains in terms of taxonomy. Accordingly, these strains may beregarded as making one group of carotenoid-producing bacteria. Thus,these bacterial strains may preferably be used in the method of thepresent invention for efficient production of astaxanthin.

According to the method of the present invention, mutant strains havingimproved astaxanthin productivity can also be used. Examples of suchmutant strains include those disclosed in JP Patent ApplicationLaid-open Publication No. 2001-95500 A and those disclosed in PatentDocument 9.

Alternatively, mutant strains having improved astaxanthin productivitycan be acquired by mutation treatment and screening. The method formutation treatment is not particularly limited as long as it inducesmutation. For example, chemical methods using a mutagen, such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate(EMS), physical methods, such as ultraviolet irradiation and X-rayirradiation, and biological methods, such as genetic recombination andtransposon, can be used. In addition, mutant strains may be thoseobtained by naturally occurring mutation. In consideration of publicacceptance or safety, it is preferable to use a microorganism that isnot a genetically recombinant microorganism.

The method for screening for a mutant strain having improved astaxanthinproductivity is not particularly limited, and may be, for example, amethod in which a mutant strain of interest is selected according to thecolor tone of a colony on an agar medium, or a method in which mutantstrains are cultured in a test tube, a flask, a fermenter, or the likeand the mutant strain of interest is selected according to carotenoidpigment analysis utilizing absorbance, high-performance liquidchromatography, thin-layer chromatography, or the like.

The steps of mutation treatment and screening may be performed once ormay be repeated twice or more such that, for example, mutant strains areobtained by mutation treatment and screening and the obtained mutantstrains are further subjected to another mutation treatment andscreening to obtain a mutant strain having improved astaxanthinproductivity.

In the method of the present invention, a mutant strain having loweredPHB (poly-β-hydroxybutyrate) producing ability may be used. For example,such a mutant strain can be induced from Paracoccus bacterial strainE-396 or A-581-1 described above or the like. It is known thatastaxanthin-producing bacteria intracellularly accumulate PHB as astorage carbon source. Accumulation of PHB causes waste of the carbonsource in a medium. Thus, it is preferable to minimize accumulation ofPHB for production cost reduction. That is, it is effective to obtain amutant strain characterized by accumulation of a small amount of PHB orno accumulation of PHB by performing mutation treatment and screening. Aspecific example of a method for obtaining a strain characterized by lowPHB production is a method in which mutation treatment is performed inthe manner described above, each mutant strain is cultured using a testtube, a flask, an agar medium, or the like, the amount of PHB isquantified, and a mutant strain characterized by low PHB production isselected.

In the method of the present invention, a mutant strain having loweredgluconic acid producing ability may also be used. For example, such amutant strain can be induced from Paracoccus bacterial strain E-396 orA-581-1 described above or the like. Production of gluconic acid resultsin waste of the carbon source in a medium corresponding to the producedgluconic acid. In addition, accumulation of a large amount of gluconicacid causes inhibition of bacterial growth or carotenoid production.Thus, it is effective to minimize gluconic acid production forcarotenoid production. A specific example of a method for obtaining astrain characterized by low gluconic acid production is a method inwhich mutation treatment is performed in the manner described above,each mutant strain is cultured in a test tube, a flask, or the like, thepH of each resulting culture liquid is measured to select mutant strainsfor which a small decrease in the pH of the culture liquid has beenconfirmed, and then the amount of gluconic acid in the culture liquid ofeach selected mutant strain is quantified to select a mutant straincharacterized by low gluconic acid production.

Mutant strains to which preferable properties have been imparted, suchas the mutant strain having improved astaxanthin productivity, themutant strain having lowered PHB producing ability, and the mutantstrain having lowered gluconic acid producing ability described above,may be separately obtained. Alternatively, mutation treatment andscreening can be repeated in order to obtain a mutant strain having twoor more such properties. It is also possible to obtain a mutant strainto which two or more properties have been simultaneously imparted bycarrying out a combination of two or more types of screening methodswith a single mutation treatment. A mutant strain having two or morepreferable properties may also be used in the method of the presentinvention.

Strain E-396 exemplified as a carotenoid-producing bacterium used in themethod of the present invention has been deposited as internationaldeposition with the International Patent Organism Depositary (IPOD), theNational Institute of Advanced Industrial Science and Technology (AIST)as described below. Strain E-396 has been deposited under the BudapestTreaty and will be irrevocably and without restriction or conditionreleased to the public upon the issuance of a patent.

International Depositary Authority:

-   -   The International Patent Organism Depositary (IPOD), the        National Institute of Advanced Industrial Science and Technology        (AIST) (the former National Institute of Bioscience and        Human-Technology, the Agency of Industrial Science and        Technology, the Ministry of International Trade and Industry)    -   Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566,        Japan

-   Identification Indication: E-396

-   Accession No.: FERM BP-4283

-   Date of the original deposit: Apr. 27, 1993

Strain A-581-1 exemplified as another carotenoid-producing bacteriumused in the method of the present invention has been deposited asinternational deposition with the above authority as described below.Strain A-581-1 has been deposited under the Budapest Treaty and will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent.

-   Identification Indication: A-581-1-   Accession No.: FERM BP-4671-   Date of the original deposit: May 20, 1994

Examples of carotenoids other than astaxanthin and canthaxanthinproduced by the method of the present invention include, but are notparticularly limited to, adonixanthin, phoenicoxanthin, β-carotene,echinenone, asteroidenone, 3-hydroxyechinenone, zeaxanthin,β-cryptoxanthin, and lycopene. Preferable examples include adonixanthinand adonirubin. One type of carotenoid or a combination of multipletypes of carotenoids may be produced according to the method of thepresent invention.

Hereinafter, a method for culturing the above-described bacteria in themethod of the present invention will be described. The term “cultureproduct” used herein is not limited to a culture liquid and thus it maycontain a solid, a semi-solid, or the like.

A medium for producing astaxanthin used for culture in the method of thepresent invention may be any medium as long as it contains biotin and itallows the growth of an astaxanthin-producing bacterium and theproduction of astaxanthin. Preferably, a medium containing a carbonsource, a nitrogen source, an inorganic salt, and if necessary, avitamin or the like is used. That is, biotin is added to a medium thatallows the growth of an astaxanthin-producing bacterium and theproduction of astaxanthin according to the method of the presentinvention.

Examples of carbon sources include: sugars such as glucose, sucrose,lactose, fructose, trehalose, mannose, mannitol, and maltose; organicacids such as acetic acid, fumaric acid, citric acid, propionic acid,malic acid, malonic acid, and pyruvic acid; alcohols such as ethanol,propanol, butanol, pentanol, hexanol, isobutanol, and glycerol; and oilsand fats such as soybean oil, rice bran oil, olive oil, corn oil, sesameoil, and linseed oil, among which glucose or sucrose is preferably used.One or more types of these carbon sources can be used. The amount of acarbon source added to a preculture medium (starting medium) differsdepending on the type of the carbon source, and may adequately beadjusted, but it is usually 1 to 100 g and preferably 2 to 50 g per 1 Lof medium. The carbon source can be added not only to the startingmedium but it may also preferably be additionally supplied duringculture in a sequential or continuous manner.

Examples of inorganic nitrogen sources include: ammonium salts such asammonium nitrate, ammonium sulfate, ammonium chloride, and ammoniumphosphate; nitrates such as potassium nitrate; ammonia; and urea. One ormore types of these inorganic nitrogen sources are used. The amount ofan added differs depending on the type of the nitrogen source and mayappropriately be adjusted, but it is usually 0.1 g to 20 g andpreferably 0.2 to 10 g per 1 L of the medium.

Examples of organic nitrogen sources include corn steep liquor(including filtrated products), pharmamedia, soybean meal, soybeanflour, peanut meal, monosodium glutamate, Distillers' solubles, anddried yeast. Among them, one or more types of organic nitrogen sourcesare used. The concentration of an organic nitrogen source added differsdepending on the type of the nitrogen source and may appropriately beadjusted, but it is usually 0 to 80 g/L and preferably 0 to 30 g/L inthe medium.

The inorganic nitrogen source and the organic nitrogen source areusually added to the starting medium; however, they may also preferablybe additionally supplied in a sequential or continuous manner.

Examples of inorganic salts include: phosphates such as potassiumdihydrogen phosphate, dipotassium hydrogen phosphate, and disodiumhydrogen phosphate; magnesium salts such as magnesium sulfate andmagnesium chloride; iron salts such as iron sulfate and iron chloride;calcium salts such as calcium chloride and calcium carbonate; sodiumsalts such as sodium carbonate and sodium chloride; manganese salts suchas manganese sulfate; cobalt salts such as cobalt chloride; copper saltssuch as copper sulfate; zinc salts such as zinc sulfate; molybdenumsalts such as sodium molybdate; nickel salts such as nickel sulfate;selenium salts such as sodium selenate; boric acid; and potassiumiodide. One or more types of these inorganic salts are used. The amountof an inorganic salt added differs depending on the type of theinorganic salt and may appropriately be adjusted, but it is usually0.0001 to 15 g per 1 L of the medium. The concentration of a phosphate,a magnesium salt, a calcium salt, a sodium salt, or an iron salt ispreferably 0.02 to 15 g/L in the medium. When a manganese salt, a cobaltsalt, a copper salt, a zinc salt, a molybdenum salt, a nickel salt, aselenium salt, boric acid, potassium iodide, or the like is added, theconcentration thereof is preferably 0.1 to 15 mg/L. The inorganic saltis usually added to the starting medium; however, it may also beadditionally supplied in a sequential or continuous manner.

Examples of vitamins other than biotin which can be used includecyanocobalamin, riboflavin, pantothenic acid, pyridoxine, thiamine,ascorbic acid, folic acid, niacin, p-aminobenzoic acid, inositol, andcholine. The proportion of a vitamin added differs depending on the typeof the vitamin and may appropriately be adjusted, but it is usually0.001 to 1000 mg and preferably 0.01 to 100 mg per 1 L of the medium.The vitamin is usually added to the starting medium; however, it mayadditionally be added in a sequential or continuous manner.

The method of the present invention has a feature of culturing anastaxanthin-producing bacterium in a medium to which biotin has beenadded. Astaxanthin of high concentration can be produced while keepingcanthaxanthin concentration at a low level by culturing anastaxanthin-producing bacterium in a medium to which biotin has beenadded.

Biotin used in the method of the present invention may be DL-biotin orD-biotin. D-biotin is preferably used. Biotin is usually added to thestarting medium; however, biotin may be added intermittently orcontinuously during culture. Alternatively, biotin may be added to thestarting medium and further added intermittently or continuously duringculture. Biotin may be mixed with a basal medium and then the medium maybe sterilized. Alternatively, biotin may separately be sterilized andthen added to a basal medium. The method for sterilizing biotin is notparticularly limited and may be heat sterilization or filtrationsterilization.

The lower limit of the concentration of biotin added to the medium isnot particularly limited, but is preferably 0.001 mg/L, more preferably0.005 mg/L, further preferably 0.01 mg/L, and particularly preferably0.02 mg/L. The upper limit of the concentration of biotin added is notparticularly limited, but is preferably 50 mg/L, more preferably 20mg/L, further preferably 10 mg/L, particularly preferably 5 mg/L, andmost preferably 2 mg/L.

An antifoamerg is preferably used in the method of the present inventionin order to prevent formation of bubbles in the culture product. Anytype of antifoamerg can be used as long as it can prevent generation ofbubbles or remove the generated bubbles with less inhibition effect onthe astaxanthin-producing bacterium. Examples of the antifoamerg includealcohol-based antifoamers, polyether-based antifoamers, ester-basedantifoamers, fatty acid-based antifoamers, silicone-based antifoamers,and sulfonic acid-based antifoamers. The amount of an antifoamerg addeddiffers depending on the type of the antifoamerg and may appropriatelybe adjusted, but it is usually 0.01 g to 10 g per 1 L of the medium.

The antifoamerg is usually added to the starting medium prior tosterilization. It may also be additionally supplied during culture in acontinuous or intermittent manner. Examples of a method for adding anantifoamerg during culture include: a method in which bubbles aredetected using a sensor so as to automatically add an antifoamerg; amethod in which an antifoamerg is added at constant time intervals usinga program timer; and a method in which an antifoamerg is mixed with acarbon source, a nitrogen source, a pH adjuster, or the like for feedingsuch that the mixture is added in response to changes in the growthrate. The antifoamerg added to the starting medium may be the same asthat added to a culture product during culture. Alternatively, differenttypes of antifoamers can be used by making use of the effects thereof.

According to the method of the present invention, pH of the medium atthe initiation of culture is adjusted to 2 to 12, preferably 6 to 9, andmore preferably 6.5 to 8.0. Preferably, pH within this range ismaintained during culture. A preferable method for maintaining pH is amethod in which pH of a culture liquid is measured online using a pHelectrode provided inside a fermenter to automatically supply alkali.Examples of pH adjusters include an aqueous sodium hydroxide solution,an aqueous potassium hydroxide solution, an aqueous sodium carbonatesolution, ammonia water, ammonia gas, an aqueous sulfuric acid solution,and a mixture thereof.

A medium used in the method of the present invention is sterilizedbefore being used for culturing a bacterium. Sterilization canappropriately be carried out by those skilled in the art. For example,the medium in a suitable container can be subjected to heatsterilization in an autoclave. Alternatively, filtration sterilizationmay be carried out using a sterilizing filter. In another case,sterilization may be carried out by jacket heating and steam injection.If a carbon source such as glucose is subjected to heat sterilizationwith other medium components, it turns brownish and thus it may beseparately sterilized. A vitamin or a minute amount of metal may besubjected to heat sterilization with a basal medium or it may beseparately sterilized in order to prevent deactivation or precipitation.

According to the method of the present invention, anastaxanthin-producing bacterium is inoculated into the medium containingbiotin prepared as described above and cultured under predeterminedconditions. Inoculation is carried out by appropriately growing thebacterial strain by seed culture using a test tube, a flask, afermenter, or the like, and adding the resulting culture product to themedium containing biotin for producing astaxanthin. The medium used forseed culture is not particularly limited and thus it may be a mediumcontaining biotin or a biotin-free medium as long as it provides goodgrowth of the astaxanthin-producing bacterium.

Culture is carried out in a suitable culture container. The culturecontainer can appropriately be selected according to the culture volume,including, for example, a test tube, a flask, a fermenter, or the like.

The temperature for culture is, for example, 15° C. to 40° C.,preferably 20° C. to 35° C., and more preferably 25° C. to 32° C.Culture is carried out under aerobic conditions within a culture periodof generally 1 day to 20 days, preferably 2 to 12 days, more preferably3 to 9 days, and particularly preferably 4 to 7 days.

Examples of aerobic conditions include shaking culture oraeration/agitation culture. Lack of oxygen would negatively influencethe growth of the astaxanthin-producing bacterium or the production ofthe carotenoid. Thus, continuous monitoring of the dissolved oxygenconcentration is preferably carried out using a dissolved oxygenelectrode.

The number of microorganisms is low immediately after the initiation ofculture, and thus the dissolved oxygen concentration is close to thesaturation concentration. However, as microorganisms grow and thus theoxygen consumption increases, the dissolved oxygen concentrationgradually decreases. Culture phases can be defined as follows: a periodbetween the initiation of culture and when the dissolved oxygenconcentration has decreased to a certain level, for example, 0 to 5 ppm,and preferably 1 to 4 ppm, which corresponds to the initial culturephase; a period between the end of the initial culture phase and whenthe concentration of produced astaxanthin has reached the maximum level,which corresponds to the intermediate phase of culture; and a periodbetween when the concentration of produced astaxanthin has reached themaximum level and the end of culture, which corresponds to the terminalphase of culture.

In order to allow the dissolved oxygen concentration to fall within thecontrol region at the earliest possible time, the aeration volume, thenumber of agitation rotations, and pressure may be set at low levels inthe initial phase of culture. Note that it is necessary to achieve theminimum necessary number of agitation rotations in order to maintain thefavorable state of mixing of a culture product. It is also necessary toachieve pressurization at the minimum necessary level in order toprevent germ contamination.

Oxygen consumption by the microorganism becomes most active in theintermediate phase of culture. Here, if aeration agitation isinsufficient, the dissolved oxygen concentration decreases to zero, thatis to say, oxygen depletion occurs. This negatively influences thegrowth of the microorganism or the production of the carotenoid. Thus,it is preferable to control the dissolved oxygen concentration toprevent oxygen depletion in the intermediate phase of culture. Thedissolved oxygen concentration can be controlled by, for example,changing the number of agitation rotations, the aeration volume,internal pressure, the oxygen concentration in an aeration gas.

According to the method of the present invention, the higher thedissolved oxygen concentration in a culture product is during culture ofan astaxanthin-producing bacterium, the lower a ratio of concentrationof produced canthaxanthin to concentration of produced astaxanthin tendsto be. Thus, the dissolved oxygen concentration in the culture productin the intermediate phase of culture is controlled preferably at 1 ppmor more, more preferably at 1.5 ppm or more, further preferably at 2 ppmor more, and particularly preferably at 2.5 ppm or more. The upper limitof the dissolved oxygen concentration in the culture product in theintermediate phase of culture that falls within the controlled range isnot particularly limited, but is preferably 8 ppm, more preferably 7ppm, further preferably 6 ppm, and particularly preferably 5 ppm.

Although the dissolved oxygen concentration in the culture product inthe intermediate phase of culture may be controlled at a given level, itis also effective to increase the dissolved oxygen concentration in astepwise or continuous manner in order to achieve high levels of theastaxanthin concentration while suppressing the canthaxanthinconcentration in the culture product. The number of steps to increasethe dissolved oxygen concentration is not particularly limited as longas it is at least one step. For example, the dissolved oxygenconcentration can be increased semi-continuously or continuously by 0.2ppm per hour for 20 hours. The lower limit of the dissolved oxygenconcentration prior to the stepwise or continuous increase of thedissolved oxygen concentration in the culture product (i.e., the initialdissolved oxygen concentration) is not limited, but is preferably 1 ppm,more preferably 1.5 ppm, and further preferably 2 ppm. The upper limitof the same is also not limited, but is preferably 3.5 ppm, morepreferably 3 ppm, and further preferably 2.5 ppm. The lower limit of thedissolved oxygen concentration following the stepwise or continuousincrease of the dissolved oxygen concentration in the culture product isnot limited, but is preferably 2.5 ppm, more preferably 3 ppm, andfurther preferably 3.5 ppm. The upper limit of the same is also notlimited, but is preferably 8 ppm, more preferably 7 ppm, furtherpreferably 6 ppm, and particularly preferably 5 ppm.

The timing at which the dissolved oxygen concentration in the cultureproduct in the intermediate phase of culture starts to be increased in astepwise or continuous manner is not particularly limited, but ispreferably 0 to 60 hours, more preferably 2 to 50 hours, furtherpreferably 4 to 40 hours, and particularly preferably 6 to 30 hours fromthe beginning of the intermediate phase of culture. The time duringwhich the dissolved oxygen concentration in a culture product starts tobe increased in a stepwise or continuous manner so as to achieve thehighest dissolved oxygen concentration is not limited. When thedissolved oxygen concentration is shifted by a single step, the highestdissolved oxygen concentration may be achieved within 1 hour. When thedissolved oxygen concentration is increased semi-continuously orcontinuously by 2 or more steps, the time during which the dissolvedoxygen concentration in the culture product starts to be increased so asto achieve the highest dissolved oxygen concentration is not limited. Inthis case, however, that time is preferably 2 to 120 hours, morepreferably 4 to 100 hours, further preferably 6 to 90 hours,particularly preferably 8 to 80 hours, and most preferably 10 to 70hours.

The dissolved oxygen concentration in the culture product can bepreferably increased or decreased during culture while carrying outsampling of the culture product as needed and analyzing the carotenoidcomposition in the intermediate phase of culture so as to adjust theratio of canthaxanthin concentration to astaxanthin concentration to adesired level. Specifically, it is effective to raise the controllinglevel of the dissolved oxygen concentration in the culture product whenthe ratio of canthaxanthin concentration to astaxanthin concentration ishigher than the desired level, while it is effective to lower thedissolved oxygen concentration in the culture product when the ratio ofcanthaxanthin concentration to astaxanthin concentration is lower thanthe same.

In the terminal phase of culture, the microorganism has a reduced levelof the activity of consuming oxygen, resulting in termination of thegrowth of the bacterium and the production of the carotenoid.Accordingly, there is no need to control the dissolved oxygenconcentration in the culture product as strictly as in the intermediatephase of culture. However, it is possible to continuously control thedissolved oxygen concentration at the same level in the late period ofthe intermediate phase of culture or to carry out culture by agitationat a constant rate with a constant aeration volume.

The ratio of canthaxanthin concentration to astaxanthin concentration inthe culture product obtained as a final product after the end of cultureby the above method is preferably 25% by mass or less, more preferably20% by mass or less, further preferably 15% by mass or less, evenfurther preferably 8% by mass or less, particularly preferably 6% bymass or less, and most preferably 4% by mass or less. The lower limit ofthe ratio of canthaxanthin concentration to astaxanthin concentration inthe culture product after the end of culture is not particularlylimited, but is preferably 0.5% by mass and more preferably 1.0% bymass. The culture product after the end of culture in which the ratio ofcanthaxanthin concentration to astaxanthin concentration is 25% by massor less can be preferably used for feed additives. In addition, theculture product in which the same is 8% by mass or less can bepreferably used for food.

A bacterium that concurrently produces astaxanthin and canthaxanthinalso produces, as a by-product, adonixanthin at the same time. A ratioof adonixanthin concentration to astaxanthin concentration in theculture product after the end of culture obtained by culture accordingto the above method is preferably 100% by mass or less, more preferably90% by mass or less, further preferably 80% by mass or less, evenfurther preferably 30% by mass or less, particularly preferably 25% bymass or less, and most preferably 20% by mass or less. The lower limitof the ratio of adonixanthin concentration to astaxanthin concentrationin the culture product after the end of culture is not particularlylimited, but is preferably 1% by mass and more preferably 5% by mass.

A bacterium used in the method of the present invention producesgluconic acid in the culture liquid. When gluconic acid is produced, itcauses waste of the carbon source in a medium corresponding to theproduced gluconic acid. When a large amount of gluconic acidaccumulates, inhibition of bacterial growth or carotenoid productionoccurs. Accordingly, it is effective to minimize gluconic acidproduction for carotenoid production. According to the method of thepresent invention, the amount of produced gluconic acid can be reducedby adding biotin to a medium. The gluconic acid concentration in theculture product obtained as a final product after the end of culture ispreferably 30 g/L or less, more preferably 20 g/L or less, furtherpreferably 10 g/L or less, and particularly preferably 5 g/L or less.The lower limit thereof is 0 g/L.

According to the method of the present invention, production of PHB(poly-β-hydroxybutyrate) can be inhibited by adding biotin to themedium. A PHB content in the culture product obtained as a final productafter the end of culture based on dry cells is preferably 30% by mass orless, more preferably 20% by mass or less, further preferably 10% bymass or less, and particularly preferably 5% by mass or less. The lowerlimit thereof is 0% by mass. In particular, the culture product in whichthe PHB content in the culture product after the end of culture based ondry cells is 30% by mass or less can be preferably used as a feedadditive.

In the method of the present invention, a carotenoid in a cultureproduct obtained by culturing an astaxanthin-producing bacterium or acarotenoid collected from a culture product can be quantified by, forexample, high performance liquid chromatography.

As described above, the culture product obtained by culturing anastaxanthin-producing bacterium may be directly used as a carotenoid.Alternatively, a culture supernatant, a cell concentrate (cellconcentrate liquid), wet cells, dry cells, cell lysate, and the like areprepared from the culture product such as a culture liquid, and they canbe used as preparations. Further, a carotenoid can be obtained from suchculture product or preparation by extraction, purification, or the like.

The culture supernatant can be prepared by subjecting the cultureproduct to centrifugation or filtration so as to remove bacterial cellsfrom the culture product. The cell concentrate (the cell concentrateliquid) can be obtained by subjecting the culture product tocentrifugation, membrane filtration concentration, or decantation. Thewet cells can be obtained by subjecting the culture product tocentrifugation or filtration. The dry cells can be obtained by dryingthe culture product, the wet cells or the cell concentrate (the cellconcentrate liquid) by a general method for drying. The dry cellscontaining a carotenoid obtained in such manner can be directly used asa feed additive.

A method for collecting a carotenoid from the above culture product orpreparation is not particularly limited in the method of the presentinvention. The method may be any method that allows efficient and stablecollection of the carotenoid. Those skilled in the art can carry outsuch a method by selecting an adequate technique from among knownextraction and purification techniques.

In addition, prior to extraction, the culture product or preparation maybe subjected to at least one treatment selected from among chemicaltreatment using an alkaline reagent or a surfactant, biochemicaltreatment using a lytic enzyme, a lipid-degrading enzyme, a protease, orthe like, and physical treatment such as ultrasonication orpulverization.

For instance, if the carotenoid is extracted from the culture product orpreparation, solvents used for extraction and washing are notparticularly limited. However, examples thereof include lower alcohols(e.g., methanol, ethanol, and isopropanol), acetone, tetrahydrofuran,methyl ethyl ketone, methyl isobutyl ketone, dichloromethane,chloroform, dimethylformamide, and dimethylsulphoxide.

If it is desired to minimize the risk of oxidation of the carotenoidduring the extraction operation, extraction can be carried out under aninert gas atmosphere such as a nitrogen gas atmosphere. It is alsopossible to select an antioxidant used for pharmaceutical agents orfoods and add it to the extraction solvent according to need.Alternatively, a combination of such treatments may be carried out.

In addition, extraction may be carried out under conditions comprisingshielding light in order to minimize the risk of degradation of thecarotenoid due to light.

The thus obtained extract can be directly used as a carotenoid and maybe further purified before use.

A method for separating the bacterium and the like from the extract(e.g., the liquid extract) obtained after the extraction operation isnot particularly limited. However, examples thereof include membranefiltration, centrifugation, and decantation.

In general, heating and/or vacuum concentration, crystallization, or thelike can be used as a method for obtaining a carotenoid precipitate fromthe extract. In addition to such method, a carotenoid pigment may beseparated by low-temperature precipitation or precipitation using anacid/alkali agent or a different salt without concentration. Forindustrial use, the extract is desirably subjected to crystallization.

If necessary, the resulting carotenoid precipitate may be subjected tosuspension/agitation for washing using a small amount of a solvent suchas lower alcohol.

A washing means is not particularly limited, while practicallypreferable methods include a method in which filtration is performedfollowing suspension/agitation and a method in which a liquid is passedthrough from above a precipitate.

The culture product, preparation, extract, or purified product obtainedin the above manner may be used alone as a carotenoid or may be mixedand used at given proportions.

EXAMPLES

Hereinafter, the present invention will be described specifically by wayof examples, although the scope of the present invention should not belimited to the following examples.

Carotenoids were quantified in the examples as described below usinghigh performance liquid chromatography (HPLC).

Two columns (Inertsil SIL-100A, 5 μm (φ): 4.6×250 mm) (GL Sciences))were used in tandem. Elution was performed by running ann-hexane/tetrahydrofuran/methanol mixture liquid (40:20:1) as a mobilephase at a rate of 1.0 mL per minute at a constant temperature aroundroom temperature. For measurement, samples dissolved in tetrahydrofuranwere 100-fold diluted with the mobile phase and 20 μL of the resultantwas injected. The column eluent was detected at a wavelength of 470 nm.Furthermore, astaxanthin (produced by Sigma) (Cat. No. A9335) was usedas a standard preparation for quantification. The concentration ofastaxanthin in the standard solution was determined according to thefollowing formula after measuring: (A): the absorbance of the standardsolution at 477 nm and; (B): the area percentage (%) of the astaxanthinpeak obtained upon HPLC analysis under the above conditions.Concentration of astaxanthin (mg/L)=A/2150×B×100

Example 1

A hundred milliliter of a medium (having the following composition:sucrose: 30 g/L; corn steep liquor: 30 g/L; potassium dihydrogenphosphate: 1.5 g/L; disodium hydrogen phosphate dodecahydrate: 3.8 g/L;calcium chloride dihydrate: 5.0 g/L; magnesium sulfate heptahydrate: 0.7g/L; and iron sulfate heptahydrate: 0.3 g/L (pH 7.2)) was poured into a500-ml cotton-plugged conical flask and sterilized in an autoclave at121° C. for 15 minutes to prepare seven flasks containing medium forseeding.

Next, 2.0 L of a medium (having the following composition: glucose: 20g/L; corn steep liquor: 30 g/L; ammonium sulfate: 0.5 g/L; potassiumdihydrogen phosphate: 2.25 g/L; disodium hydrogen phosphatedodecahydrate: 5.7 g/L; calcium chloride dihydrate: 0.1 g/L; magnesiumsulfate heptahydrate: 0.5 g/L; iron sulfate heptahydrate: 5 g/L; and analcohol-based antifoamerg: 0.5 g/L) was poured into a 5-L fermenter.Seven fermentersg were prepared in such manner. D-biotin was added tothe fomenters to result in concentrations of 0, 0.001, 0.01, 0.1, 1.0,10, and 50 mg/L, respectively, and each resultant was sterilized in anautoclave at 121° C. for 30 minutes.

A platinum loopful of Paracoccus carotinifaciens strain E-396 (FERMBP-4283) was inoculated into the medium for seeding in each flaskprepared above, followed by rotary shaking culture at 100 rpm and 28° C.for 2 days. Subsequently, the resulting culture liquids (80 mL each)were separately introduced into the fomenters for aerobic culture at 28°C. with an aeration volume of 1 vvm for 120 hours. pH was continuouslycontrolled with 15% ammonia water so as to be maintained at 7.2 duringculture. Glucose (30 g) was added on the 1st, 2nd, 3rd, and 4th days ofculture to prevent depletion of glucose. In addition, the number ofagitation rotations was changed (the lowest number of agitationrotations: 200 rpm) to maintain the dissolved oxygen concentration ineach culture liquid at 2 ppm in the intermediate phase of culture. Oncebubble formation was detected with a bubble sensor, an alcohol-basedantifoamerg was automatically added to prevent bubble formation.

The carotenoid concentration, the gluconic acid concentration, and thePHB content based on dry cells in each culture liquid at the end ofculture were determined. Table 1 shows the results. It was found thatthe ratio of concentration of canthaxanthin to concentration ofastaxanthin was lower in each experimental plot to which biotin had beenadded at 0.001 to 50 mg/L than that in a plot to which no biotin hadbeen added.

TABLE 1 Biotin concentration mg/L 0 0.001 0.01 0.1 1 10 50 Canthaxanthinmg/L 5.2 3.9 3.3 3.3 3.0 2.7 2.5 Astaxanthin mg/L 15.2 15.7 16.5 17.015.9 14.3 14.1 Adonixanthin mg/L 6.3 6.6 6.7 6.9 6.8 7.3 7.2Canthaxanthin/ 34.2 24.8 20.0 19.4 18.9 18.9 17.7 Astaxanthin mass %Adonixanthin/ 41.4 42.0 40.6 40.6 42.8 51.0 51.1 Astaxanthin mass %Gluconic acid g/L 45 28 19 14 13 13 10 PHB mass % 35 29 24 25 22 21 23

Example 2

A hundred milliliter of a medium (having the following composition:sucrose: 30 g/L; corn steep liquor: 30 g/L; potassium dihydrogenphosphate: 1.5 g/L; disodium hydrogen phosphate dodecahydrate: 3.8 g/L;calcium chloride dihydrate: 5.0 g/L; magnesium sulfate heptahydrate: 0.7g/L; and iron sulfate heptahydrate: 0.3 g/L (pH 7.2)) was poured into a500-ml cotton-plugged conical flask and sterilized in an autoclave at121° C. for 20 minutes to prepare eight flasks containing medium forseeding.

Next, 2.0 L of a medium (having the following composition: sucrose: 40g/L; corn steep liquor: 30 g/L; ammonium sulfate: 0.5 g/L; potassiumdihydrogen phosphate: 2.25 g/L; disodium hydrogen phosphatedodecahydrate: 5.7 g/L; calcium chloride dihydrate: 0.1 g/L; magnesiumsulfate heptahydrate: 0.5 g/L; iron sulfate heptahydrate: 5 g/L; and analcohol-based antifoamerg: 0.5 g/L) was poured into a 5-L fermenter.Eight tanks were prepared in such manner. D-biotin was added to eachfermenter to result in 0.1 mg/L, and each resultant was sterilized in anautoclave at 121° C. for 30 minutes.

A platinum loopful of Paracoccus carotinifaciens strain E-396 (FERMBP-4283) was inoculated into the medium for seeding in each flaskprepared above, followed by rotary shaking culture at 100 rpm and 28° C.for 2 days. Subsequently, the resulting culture liquids (80 mL each)were separately introduced into the fomenters for aerobic culture at 28°C. with an aeration volume of 1 vvm for 120 hours. pH was continuouslycontrolled with 15% ammonia water so as to be maintained at 7.2 duringculture. Sucrose (30 g) was added on the 1st, 2nd, 3rd, and 4th days ofculture to prevent depletion of sucrose. In addition, the number ofagitation rotations was changed (the lowest number of agitationrotations: 200 rpm) to maintain the dissolved oxygen concentrations inthe culture liquids at 0.5, 1, 2, 3, 4, 5, 6, and 7 ppm in theintermediate phase of culture. Once bubble formation was detected with abubble sensor, an alcohol-based antifoamerg was automatically added toprevent bubble formation.

The carotenoid concentration, the gluconic acid concentration, and thePHB content based on dry cells in each culture liquid at the end ofculture were determined. Table 2 shows the results. It was found thatthe ratio of canthaxanthin concentration to astaxanthin concentrationtended to decrease as the dissolved oxygen concentration was increased,and that the ratio can be reduced to 1.4%.

For comparison, a similar experiment was performed using a biotin-freemedium. Table 3 shows the results. The ratio of canthaxanthinconcentration to astaxanthin concentration was able to be reduced to 9%at most.

TABLE 2 Addition of 0.1 mg/L biotin Dissolved oxygen concentration ppm0.5 1 2 3 4 5 6 7 Canthaxanthin 7.3 3.3 1.6 1.2 0.6 0.3 0.2 0.1 mg/LAstaxanthin 8.3 13.3 20.2 20.9 15.8 11.3 9.6 7.0 mg/L Adonixanthin 1.42.3 3.9 5.1 6.1 8.2 8.8 11.4 mg/L Canthaxanthin/ 88.0 24.8 7.9 5.7 3.82.7 2.1 1.4 Astaxanthin mass % Adonixanthin/ 16.9 17.3 19.3 24.4 38.672.6 91.7 163 Astaxanthin mass % Gluconic acid 24 19 17 14 16 11 10 9g/L PHB mass % 25 19 18 17 14 20 13 17

TABLE 3 Without addition of biotin Dissolved oxygen concentration ppm0.5 1 2 3 4 5 6 7 Canthaxanthin 6.7 4.1 3.2 2.2 1.2 0.7 0.5 0.4 mg/LAstaxanthin 3.0 7.8 12.3 13.3 10.9 7.8 5.5 4.2 mg/L Adonixanthin 0.6 1.73.4 5.5 6.8 6.5 7.3 8.7 mg/L Canthaxanthin/ 223 52.6 26.0 16.5 11.0 9.09.1 9.5 Astaxanthin mass % Adonixanthin/ 20.0 21.8 27.6 41.4 62.4 83.3133 207 Astaxanthin mass % Gluconic acid 56 47 33 44 35 36 31 31 g/L PHBmass % 44 47 29 34 27 27 36 32

Example 3

Paracoccus carotinifaciens strain E-396 (FERM BP-4283) was subjected tomutation treatment with N-methyl-N′-nitro-N-nitrosoguanidine, and acolony taking on a strong red color was selected. The PHB concentrationand the carotenoid concentration in the culture liquid of the selectedstrain were determined, and the LP-26 mutant strain having low PHBproducing ability and high astaxanthin producing ability was selected.

A hundred milliliter of a medium (having the following composition:sucrose: 30 g/L; corn steep liquor: 30 g/L; potassium dihydrogenphosphate: 1.5 g/L; disodium hydrogen phosphate dodecahydrate: 3.8 g/L;calcium chloride dihydrate: 5.0 g/L; magnesium sulfate heptahydrate: 0.7g/L; and iron sulfate heptahydrate: 0.3 g/L; (pH 7.2)) was poured into a500-ml cotton-plugged conical flask and sterilized in an autoclave at121° C. for 20 minutes to prepare a flask containing the medium forseeding.

Next, 2.0 L of a medium (having the following composition: glucose: 30g/L; corn steep liquor: 30 g/L; ammonium sulfate: 0.5 g/L; potassiumdihydrogen phosphate: 2.25 g/L; disodium hydrogen phosphatedodecahydrate: 5.7 g/L; calcium chloride dihydrate: 0.1 g/L; magnesiumsulfate heptahydrate: 0.5 g/L; iron sulfate heptahydrate: 5 g/L;L-monosodium glutamate monohydrate: 6 g/L; and an alcohol-basedantifoamerg: 0.5 g/L) was poured to a 5-L fermenter. Eight fomenterswere prepared in such manner. D-biotin was added to each fermenter toresult in 0.1 mg/L, and each resultant was sterilized in an autoclave at121° C. for 30 minutes.

A platinum loopful of Paracoccus bacterial strain LP-26 selected abovewas inoculated into the medium for seeding in the flask as describedabove, followed by rotary shaking culture at 100 rpm and 28° C. for 2days. The resulting culture liquids (80 mL each) were separatelyintroduced into the fomenters for aerobic culture at 28° C. with anaeration volume of 1 vvm for 140 hours. The pH was continuouslycontrolled with 15% ammonia water so as to be maintained at 7.2 duringculture. Glucose (50 g) was added on the 1st, 2nd, 3rd, and 4th days ofculture to prevent depletion of glucose. In addition, the number ofagitation rotations was changed (the lowest number of agitationrotations: 100 rpm) to maintain the dissolved oxygen concentrations inthe culture liquids at 0.5, 1, 2, 3, 4, 5, 6, and 7 ppm in theintermediate phase of culture. Once bubble formation was detected with abubble sensor, an alcohol-based antifoamerg was automatically added toprevent bubble formation.

The carotenoid concentration, the gluconic acid concentration, and thePHB content based on dry cells in each culture liquid at the end ofculture were determined. Table 4 shows the results. It was found thatthe ratio of canthaxanthin concentration to astaxanthin concentrationtended to decrease as the dissolved oxygen concentration was increased,and that the ratio can be reduced to 1.6%.

For comparison, a similar experiment was performed using a biotin-freemedium while controlling the dissolved oxygen concentration at 2 ppm inthe intermediate phase of culture. Table 5 shows the results. The ratioof canthaxanthin concentration to astaxanthin concentration was 8% inthe medium to which biotin had been added while controlling thedissolved oxygen concentration at 2 ppm. On the other hand, it was ashigh as 26.8% in the biotin-free medium.

TABLE 4 Addition of 0.1 mg/L biotin Dissolved oxygen concentration ppm0.5 1 2 3 4 5 6 7 Canthaxanthin 243 122 59 52 24 10 6 4 mg/L Astaxanthin301 499 741 746 577 401 315 244 mg/L Adonixanthin 45 73 130 201 255 259309 332 mg/L Canthaxanthin/ 80.7 24.4 8.0 7.0 4.2 2.5 1.9 1.6Astaxanthin mass % Adonixanthin/ 15.0 14.6 17.5 26.9 44.2 64.6 98.1 136Astaxanthin mass % Gluconic acid 26 17 19 15 15 16 14 10 g/L PHB mass %12 10 9 6 1 3 0 6

TABLE 5 Without addition of biotin Dissolved oxygen concentration ppm 2Canthaxanthin mg/L 132 Astaxanthin mg/L 493 Adonixanthin mg/L 144Canthaxanthin/Astaxanthin mass % 26.8 Adonixanthin/Astaxanthin mass %29.2 Gluconic acid g/L 35 PHB mass % 22

Next, experiments for shifting the dissolved oxygen concentration in astepwise or continuous manner in the intermediate phase of culture wereperformed under the four different conditions described below, with thecondition that D-biotin was added to result in 0.1 mg/L and the aboveconditions other than the conditions for the dissolved oxygenconcentration were employed.

(Shift Condition 1)

The initial dissolved oxygen concentration in the intermediate phase ofculture was controlled at 2 ppm and maintained at 2 ppm for 40 hours.Then, the dissolved oxygen concentration was shifted to 2.5 ppm andmaintained at 2.5 ppm until the end of culture. Specifically, thedissolved oxygen concentration was allowed to spontaneously decreasefrom the saturation concentration to 2 ppm within 0 to 8 hours after theinitiation of culture, and controlled at 2 ppm within 8 to 48 hours and2.5 ppm within 48 to 140 hours.

(Shift Condition 2)

The initial dissolved oxygen concentration in the intermediate phase ofculture was controlled at 1 ppm, the level of 1 ppm was maintained for 8hours, the controlled concentration was increased by 0.1 ppm every anhour such that the dissolved oxygen concentration was increased to 5 ppmwithin 40 hours, following which the level of 5 ppm was maintained untilthe end of culture. Specifically, the dissolved oxygen concentration wasallowed to spontaneously decrease from the saturation concentration to 1ppm within 0 to 9 hours after the initiation of culture, controlled at 1ppm within 9 to 17 hours, increased by 0.1 ppm every an hour from 1 ppmto 5 ppm within 17 to 57 hours, and controlled at 5 ppm within 57 to 140hours.

(Shift Condition 3)

The initial dissolved oxygen concentration in the intermediate phase ofculture was controlled at 3 ppm and maintained at 3 ppm for 50 hours.Then, the dissolved oxygen concentration was shifted to 3.5 ppm andmaintained at 3.5 ppm until the end of culture. Specifically, thedissolved oxygen concentration was allowed to spontaneously decreasefrom the saturation concentration to 3 ppm within 0 to 7 hours after theinitiation of culture, and controlled at 3 ppm within 7 to 57 hours and3.5 ppm within 57 to 140 hours.

(Shift Condition 4)

The initial dissolved oxygen concentration in the intermediate phase ofculture was controlled at 2 ppm, the level of 2 ppm was maintained for 4hours, the controlled concentration was increased by 0.1 ppm every twohours such that the dissolved oxygen concentration was increased to 7ppm within 100 hours, following which the level of 7 ppm was maintaineduntil the end of culture. Specifically, the dissolved oxygenconcentration was allowed to spontaneously decrease from the saturationconcentration to 2 ppm within 0 to 8 hours after the initiation ofculture, controlled at 2 ppm within 8 to 12 hours, increased by 0.1 ppmevery two hours from 2 ppm to 7 ppm within 12 to 112 hours, andcontrolled at 7 ppm within 112 to 140 hours.

Culture was carried out under the above four different conditions. Thecarotenoid concentration, the gluconic acid concentration, and the PHBcontent based on dry cells in each culture liquid at the end of culture(140 hours after the initiation of culture) were determined. Table 6shows the results. The results were compared with those obtained bycontrolling the dissolved oxygen concentration at given levels (Table4). Accordingly, it was found that a culture liquid having a highconcentration of produced astaxanthin and a lower ratio of producedcanthaxanthin can be obtained under any of the above conditions.

TABLE 6 Addition of 0.1 mg/L biotin Condition for shifting dissolvedoxygen concentration Condition Condition Condition Condition 1 2 3 4Canthaxanthin mg/L 154 166 39 41 Astaxanthin mg/L 796 863 752 800Adonixanthin mg/L 156 162 214 212 Canthaxanthin/ 19.3 19.2 5.2 5.1Astaxanthin mass % Adonixanthin/ 19.6 18.8 28.5 26.5 Astaxanthin mass %Gluconic acid g/L 8 11 13 6 PHB mass % 3 2 5 0

Example 4

Paracoccus carotinifaciens strain E-396 (FERM BP-4283) was subjected tomutation treatment with N-methyl-N′-nitro-N-nitrosoguanidine, and acolony taking on a strong red color was selected. The selected strainwas cultured in a test tube. The mutant strain was selected with thecondition that a decrease in pH of the culture liquid was small and theculture liquid took on a strong red color. The gluconic acidconcentration and the carotenoid concentration in the test tube cultureliquid of the selected mutant strain were determined, and the LG-7mutant strain having low gluconic acid producing ability and highastaxanthin producing ability was selected.

A hundred milliliter of a medium (having the following composition:sucrose: 30 g/L; pharmamedia: 10 g/L; potassium dihydrogen phosphate:0.8 g/L; dipotassium hydrogen phosphate: 4.2 g/L; calcium chloridedihydrate: 1 g/L; magnesium sulfate heptahydrate: 12 g/L; and ironsulfate heptahydrate: 1 g/L (pH 7.2)) was poured into a 500-mlcotton-plugged conical flask and sterilized in an autoclave at 121° C.for 20 minutes to prepare a flask containing the medium for seeding.

Next, 2.0 L of a medium (having the following composition: sucrose: 30g/L; pharmamedia: 20 g/L; ammonium sulfate: 1.5 g/L; potassiumdihydrogen phosphate: 1.5 g/L; disodium hydrogen phosphatedodecahydrate: 3.8 g/L; calcium chloride dihydrate: 0.1 g/L; magnesiumsulfate heptahydrate: 4.5 g/L; iron sulfate heptahydrate: 5 g/L;L-monosodium glutamate monohydrate: 6 g/L; a silicone-based antifoamerg:1 g/L) was poured into a 5-L fermenter. Two fomenters were prepared insuch manner. D-biotin was added to each fermenter to result in 1 mg/L,and each resultant was sterilized in an autoclave at 121° C. for 30minutes.

A platinum loopful of the Paracoccus LG-7 strain selected above wasinoculated into the medium for seeding in the flask prepared above,followed by rotary shaking culture at 100 rpm and 28° C. for 3 days. Theculture liquids (80 mL each) were separately introduced into thefomenters for aerobic culture at 28° C. with an aeration volume of 1 vvmfor 120 hours. pH was continuously controlled with 15% ammonia water soas to be maintained at 7.2 during culture. Sucrose (40 g) was added onthe 1st, 2nd, 3rd, and 4th days of culture to prevent depletion ofsucrose. In addition, the number of agitation rotations was changed (thelowest number of agitation rotations: 200 rpm) to control theconcentration of dissolved oxygen in the culture liquid under thefollowing two conditions.

(Shift Condition 5)

The initial dissolved oxygen concentration in the intermediate phase ofculture was controlled at 2.5 ppm and maintained at 2.5 ppm for 35hours. Then, the dissolved oxygen concentration was shifted to 3 ppm andmaintained at 3 ppm until the end of culture. Specifically, thedissolved oxygen concentration was allowed to spontaneously decreasefrom the saturation concentration to 2.5 ppm within 0 to 8 hours afterthe initiation of culture, and controlled at 2.5 ppm within 8 to 43hours and 3 ppm within 43 to 120 hours.

(Shift Condition 6)

The initial dissolved oxygen concentration in the intermediate phase ofculture was controlled at 3.5 ppm, the level of 3.5 ppm was maintainedfor 4 hours, the controlled concentration was increased by 0.1 ppm everyfour hours such that the dissolved oxygen concentration was increased to5 ppm within 60 hours, following which the level of 5 ppm was maintaineduntil the end of culture. Specifically, the dissolved oxygenconcentration was allowed to spontaneously decrease from the saturationconcentration to 3.5 ppm within 0 to 7 hours after the initiation ofculture, controlled at 3.5 ppm within 7 to 11 hours, increased by 0.1ppm every four hours from 3.5 ppm to 5 ppm within 11 to 71 hours, andcontrolled at 5 ppm within 71 to 120 hours.

Culture was carried out under the above two different conditions. Thecarotenoid concentration, the gluconic acid concentration, and the PHBcontent based on dry cells in each culture liquid at the end of culture(120 hours after the initiation of culture) were determined. Table 7shows the results. Table 7 also shows the culture results obtained bycontrolling the dissolved oxygen concentration in the intermediate phaseof culture and that in the terminal phase of culture at a constant levelof 4 ppm without the addition of biotin for comparison.

TABLE 7 Biotin concentration 1 mg/L 1 mg/L 0 Dissolved oxygen Condition5 Condition 6 4 ppm concentration condition (constant) Canthaxanthinmg/L 61 25 40 Astaxanthin mg/L 607 429 414 Adonixanthin mg/L 133 252 258Canthaxanthin/ 10.0 5.8 9.7 Astaxanthin mass % Adonixanthin/ 21.9 58.762.4 Astaxanthin mass % Gluconic acid g/L 3 0 7 PHB mass % 17 18 35

Example 5

A hundred milliliter of a medium (having the following composition:sucrose: 20 g/L; corn steep liquor: 5 g/L; potassium dihydrogenphosphate: 0.54 g/L; dipotassium hydrogen phosphate: 2.78 g/L; calciumchloride dihydrate: 5 g/L; magnesium sulfate heptahydrate: 0.7 g/L; andiron sulfate heptahydrate: 3 g/L (pH 7.2)) was poured into a 500-mlcotton-plugged conical flask and sterilized by autoclave at 121° C. for20 minutes to prepare a flask containing the medium for seeding.

Next, 2.0 L of a medium (having the following composition: glucose: 40g/L; corn steep liquor: 30 g/L; ammonium sulfate: 0.5 g/L; potassiumdihydrogen phosphate: 2.25 g/L; disodium hydrogen phosphatedodecahydrate: 5.7 g/L; calcium chloride dihydrate: 0.1g/L; magnesiumsulfate heptahydrate: 0.5 g/L; iron sulfate heptahydrate: 5 g/L;L-monosodium glutamate monohydrate: 6 g/L; and an alcohol-basedantifoamerg: 0.5 g/L) was poured into a 5-L fermenter. D-biotin wasadded to the fermenter to result in a concentration of 0.1 mg/L, and theresultant was sterilized in an autoclave at 121° C. for 30 minutes.

A platinum loopful of Paracoccus bacterial strain A-581-1 (FERM BP-4671)was inoculated into the medium for seeding in the flask prepared above,followed by rotary shaking culture at 150 rpm and 27° C. for 2 days. Theculture liquid (80 mL) was poured into the fermenter for aerobic cultureat 28° C. with an aeration volume of 1 vvm for 120 hours. pH wascontinuously controlled with 15% ammonia water so as to be maintained at7.2 during culture. Glucose (30 g) was added on the 1st, 2nd, 3rd, and4th days of culture to prevent depletion of glucose. In addition, thenumber of agitation rotations was changed (the lowest number ofagitation rotations: 200 rpm) to control the concentration of dissolvedoxygen in the culture liquid at 2 ppm in the intermediate phase ofculture.

The carotenoid concentration, the gluconic acid concentration, and thePHB content based on dry cells at the end of culture were determined.Table 8 shows the results. Table 8 also shows the culture resultsobtained by controlling the dissolved oxygen concentration in theintermediate phase of culture at a constant level of 2 ppm without theaddition of biotin for comparison.

TABLE 8 Biotin concentration 0.1 mg/L 0 Dissolved oxygen concentration 2ppm (constant) 2 ppm (constant) condition Canthaxanthin mg/L 1.7 3.3Astaxanthin mg/L 7.5 6.9 Adonixanthin mg/L 1.8 2.0Canthaxanthin/Astaxanthin mass % 22.7 47.8 Adonixanthin/Astaxanthin mass% 24.0 29.0 Gluconic acid g/L 17 40 PHB mass % 18 29

Example 6

Paracoccus bacterial strain A-581-1 (FERM BP-4671) was subjected toultraviolet irradiation for mutation treatment. Accordingly, a colonytaking on a strong red color was selected. Carotenoids in the cultureliquid of the selected strain were analyzed. Thus, the K-185 mutantstrain having improved astaxanthin productivity was selected.

A hundred milliliter of a medium (having the following composition:sucrose: 30 g/L; corn steep liquor: 30 g/L; potassium dihydrogenphosphate: 1.5 g/L; disodium hydrogen phosphate dodecahydrate: 3.8 g/L;calcium chloride dihydrate: 5 g/L; magnesium sulfate heptahydrate: 0.7g/L; and iron sulfate heptahydrate: 0.3 g/L; (pH 7.2)) was poured into a500-ml cotton-plugged conical flask and sterilized by autoclave at 121°C. for 20 minutes to prepare a flask containing the medium for seeding.

Next, 2.0 L of a medium (having the following composition: glucose: 30g/L; soybean meal: 20 g/L; ammonium sulfate: 1.5 g/L; potassiumdihydrogen phosphate: 1.5 g/L; disodium hydrogen phosphatedodecahydrate: 3.8 g/L; calcium chloride dihydrate: 5 g/L; magnesiumsulfate heptahydrate: 0.7 g/L; iron sulfate heptahydrate: 0.6 g/L;L-monosodium glutamate monohydrate: 6 g/L; and an ester-basedantifoamerg: 0.2 g/L) was poured into a 5-L fermenter. D-biotin wasadded to the fermenter to result in a concentration of 1 mg/L, and theresultant was sterilized in an autoclave at 121° C. for 30 minutes.

A platinum loopful of Paracoccus bacterial strain K-185 selected abovewas inoculated into the medium for seeding in the flask prepared above,followed by rotary shaking culture at 150 rpm and 27° C. for 2 days. Theculture liquid (80 mL) was added to the fermenter for aerobic culture at28° C. with an aeration volume of 1 vvm for 120 hours. pH wascontinuously controlled with 15% ammonia water so as to be maintained at7.2 during culture. Glucose (30 g) was added on the 1st, 2nd, 3rd, and4th days of culture to prevent depletion of glucose. In addition, thenumber of agitation rotations was changed (the lowest number ofagitation rotations: 200 rpm) to control the concentration of dissolvedoxygen in the culture liquid at 3.5 ppm in the intermediate phase ofculture.

The carotenoid concentration, the gluconic acid concentration, and thePHB content based on dry cells at the end of culture were determined.Table 9 shows the results. Table 9 also shows the culture resultsobtained by controlling the dissolved oxygen concentration in theintermediate phase of culture at 3.5 ppm without the addition of biotinfor comparison.

TABLE 9 Biotin concentration 1 mg/L 0 Dissolved oxygen concentration 3.5ppm 3.5 ppm condition (constant) (constant) Canthaxanthin mg/L 2.2 6.0Astaxanthin mg/L 39.0 35.2 Adonixanthin mg/L 29.6 28.9Canthaxanthin/Astaxanthin mass % 5.6 17.0 Adonixanthin/Astaxanthin mass% 75.9 82.1 Gluconic acid g/L 19 38 PHB mass % 15 33

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

The invention claimed is:
 1. A method for producing carotenoidscomprising: 1) culturing a bacterial strain in a culture mediumcontaining biotin at a concentration of 0.02 mg/L to 50 mg/L by additionof biotin to the culture medium, wherein the bacterial strain is amember of the genus Paracocrus and concurrently produces astaxanthin,canthaxanthin and adonixanthin; 2) increasing the concentration ofdissolved oxygen in the culture medium in a stepwise or continuousmanner during the intermediate phase of culture, wherein theconcentration of dissolved oxygen in the culture medium at the beginningof the intermediate phase of culture before increasing the concentrationis 1.5 ppm to 2.5 ppm, and the concentration of dissolved oxygen in theculture medium at the end of the intermediate phase of culture afterincreasing the concentration is 3 ppm to 6 ppm, thereby producing acarotenoid-containing culture broth in which the ratio of theconcentration of canthaxanthin to the concentration of astaxanthin inthe culture broth at the end of the culture period is 8% to 15% and theratio of the concentration of adonixanthin to the concentration ofastaxanthin in the culture broth at the end of the culture period is 25%or less; and 3) collecting the carotenoids from thecarotenoid-containing culture broth.
 2. The method of claim 1, whereinthe concentration of gluconic acid in the carotenoid-containing culturebroth at the end of the culture period is 30 g/L or less.
 3. The methodof claim 1, wherein a poly-β-hydroxybutyrate accumulation in thecarotenoid-containing culture broth at the end of the culture period is30% by mass or less based on dry cells.
 4. The method of claim 1,wherein the bacterial strain is strain E-396 (FERM BP-4283) or strainA-581-1 (FERM BP-4671.
 5. The method of claim 1, wherein the bacterialstrain is Paracoccus carotinifaciens.
 6. The method of claim 1, whereinthe concentration of dissolved oxygen in the culture medium at thebeginning of the intermediate phase of culture before increasing theconcentration is 2 ppm to 2,5 ppm.
 7. The method of claim 1, wherein theconcentration of dissolved oxygen in the culture medium at the beginningof the intermediate phase of culture before increasing the concentrationis 2 ppm.
 8. The method of claim 1, wherein the concentration ofdissolved oxygen in the culture medium at the end of the intermediatephase of culture after increasing the concentration is 3 ppm to 5 ppm.9. The method of claim 1, wherein the concentration of dissolved oxygenin the culture medium at the end of the intermediate phase of cultureafter increasing the concentration is 3 ppm.
 10. The method of claimwherein the concentration of dissolved oxygen in the culture mediumstarts to be increased 2 to 50 hours from the beginning of theintermediate phase of culture.